Method of treating of demyelinating diseases or conditions

ABSTRACT

N-(Pyridinyl)-1H-indol-1-amines of formula I provide a unique combination of blocking properties for both the potassium and sodium channels. These compounds are useful for the treatment of Demyelinating Diseases and Conditions such as Multiple Sclerosis, Spinal Cord Injury, Traumatic Brain Injury and Stroke. The compounds are also useful for Stroke Rehabilitation, the treatment of Bladder Irritation and Dysfunction, and the treatment of Neuropathic Pain and Chemokine-Induced Pain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/770,656 filed on Feb. 3, 2004 now abandoned, which is a divisional ofU.S. application Ser. No. 10/076,191 filed on Feb. 14, 2002 now U.S.Pat. No. 6,967,210, which claims the benefit of U.S. ProvisionalApplication No. 60/268,846, filed Feb. 15, 2001, and claims priority toGB 0119435.6, filed Aug. 9, 2001, which are hereby incorporated byreference herein in their entireties.

BACKGROUND OF THE INVENTION

Multiple sclerosis (MS) is a degenerative and inflammatory neurologicaldisease that affects the central nervous system, and is associated withformation of neuronal plaques and impaired neuronal conduction due todemyelination (loss of myelin). Similarly, extensive demyelination iscommonly reported in spinal cord trauma and stroke (Bunge et al, 1993;Blight and DeCrescito, 1986; Pendlebury et al, 2000).

Basic research into the physiology of the action potential propagationin myelinated fibers showed that conduction block in demyelinated fiberswas partly due to the appearance of aminopyridine-sensitive potassiumchannels in areas of myelin loss (Bever 1996).

Action potentials propagate along normal myelinated nerve fibers by aprocess of salutatory conduction, which results from a sodium currentgenerated by the opening of voltage-sensitive sodium channels at thenode of Ranvier. Thus, at the onset of electrical stimulation, sodium(Na⁺) ions enter the neuron, causing the neuron to become morepositively charged. When the positive nature of the neuron approaches acritical level, “depolarization” occurs. Depolarization allows apositive core of ions to flow down the neuron, along the axon and to thenerve ending. For the neuron to “reset” itself, the excess positivecharge must be dissipated. This is done via the outflow of potassiumions (hereinafter “K⁺”) through potassium channels. When myelin isdisrupted, voltage-sensitive potassium channels that open duringdepolarization appear on the axolemma. The potassium current, flowingopposite to the sodium current, decreases action potential amplitude andduration, contributing to conduction failure by decreasing the distaleffective current densities. These conduction deficits are associatedwith disabling symptoms, including muscle weakness. By blocking theoutflow of K⁺ through potassium channels, the neuron remains depolarizedlonger and is more easily restimulated. Thus, potassium channel blockersare believed to be useful in the treatment of diseases and conditionswhich impair action potential transmission such as MS, Traumatic BrainInjury (hereinafter “TBI”) and Spinal Cord Injury (hereinafter “SCI”).

Potassium channel blockers, such as 4-amino pyridine (hereinafter“4-AP”), increase action potential duration and amplitude indemyelinated fibers and improve action potential propagation in vitro(Bostock et al, 1978; 1981; Targ and Kocsis, 1985; 1986; Shi and Blight,1997), facilitate neurotransmitter release (Bostock et al, 1981; Hirshand Quandt, 1993; Sherratt et al, 1980), and potentiate musclecontractility (Agoston et al, 1982; Savage et al, 1985). Theseobservations suggested that potassium channel blockers, such as 4-AP,could restore conduction in demyelinated fibers in MS patients.Subsequent clinical trial results lend further support the propositionthat aminopyridine treatment may improve symptoms in some MS patients(Jones et al 1983; Stefoski et al, 1987; Davis et al, 1990; van Diemenet al, 1992; Bever et al, 1994; Schwid et al, 1997).

4-AP has also been disclosed to be effective in the treatment ofneurological conditions including SCI, reduction of chronic pain andspasticity in SCI patients, Alzheimer's disease, post-polio syndrome,myasthenia gravis, Huntington's disease, age-related memory disorders,post-traumatic, post-stroke or post-toxic syndromes affecting memory orcognition, and dysautonomia (Wurtman R J and Buyukuysal R, 1989;Hansebout R R and Blight A, 1996; Hansebout R R and Blight A 1994).Clinical studies for the use of Fampridine-SR in long-term spinal cordinjured patients have begun (Potter et al, 1998a,b) notwithstandingsafety concerns surrounding use of 4-AP in the general patientpopulation (Multiple Sclerosis, Cognos Study #51, Decisions Resources,October, 1999; pp77–8). Several studies have shown that single doses of4-AP can restore some function in SCI patients when administered oneyear or longer after injury (Potter et al, 1998a,b; Qiao et al, 1997;Hayes et al, 1993; 1994). Positive effects after chronic dosing havealso been reported. Clinically significant functional improvements wereobserved in 16 out of 16 patients after 3 months of daily oral dosingwith 30 mg/kg 4-AP in patients with SCI of 2 years or more. Somepatients previously classified as having complete injury werereclassified to incomplete injury level (Segal et al, 1999). Allpatients showed some degree of improvement in at least some type ofneurolgic or pulmonary function after 3 months of daily oral treatmentwith 4-AP (30 mg/day, or approximately 0.5 mg/kg). A lower dose was notactive.

As previously stated, 4-AP blocks potassium channels, effectivelyprolonging the action potential. Unfortunately, this mechanism by whichpotassium channel blockers can improve symptoms associated with diseasesand conditions which impair action potential transmission can also leadto epileptic-like activity. Indeed, 4-AP is a recognized convulsiveagent in animals and humans. Therefore, the usefulness of 4-AP as atherapeutic agent for MS, TBI and SCI is tempered by its pro-convulsantliability and other undesirable side effects. Restlessness, confusion,and generalized tonic-clonic seizures have been reported at doses higherthan 0.8 mg/kg (Ball et al, 1979; Bever et al, 1994). Van Diemen et al(1993) reported that magnitude of improvement in MS patients (defined byimprovement in smooth pursuit gain) was significantly related to 4-APserum level, (33–75 ng/ml necessary for significant improvement afteroral administration). However, side effects (paresthesia/dysestheia,dizziness/light-headedness, and even gait instability) were observed atthe same doses. In another human study, Bever et al (1994) reported agrand mal seizure at a serum level of 104 ng/ml. Both groups ofinvestigators suggested that higher dosages and serum levels would belikely to produce greater improvements in those MS patients whichresponded to lower doses of 4-AP. Thus, the degree of efficacy with 4-APis dose- and side effect-limited.

Concern about the side-effects associated with higher 4-AP serum levelshas led to the development of sustained release formulations(Fampridine-SR) (Masterson J G and Myers M, 1994; 1996a; 1996b).Fampridine-SR is currently in Phase 2 clinical studies for MS. Patientsin prior clinical studies of Fampridine-SR have shown improvement in avariety of functions. Depending on the individual, these improvementshave included enhanced bladder, bowel, and sexual function, increasedease of movement and sensation, and reduced muscle spasticity, fatigueand chronic pain.

Another approach to eliminating the undesirable side effects associatedwith 4-AP involves coadministration of 4-AP and voltage dependent sodiumchannel blockers. Sodium (Na⁺) channel blockers block the inflow of Na⁺ions and reduce the susceptibility of the neuron to depolarization. Thiseffectively reduces neuronal excitability. Indeed, it has been reportedthat coadministration of voltage-dependent sodium channel blockers and4-AP prevents 4-AP-induced convulsions in mice (Yamaguchi and Rogawski,1992). 4-AP has no sodium channel blocking properties.

The compounds used in the methods claimed herein can be synthesized viaprocedures disclosed in U.S. Pat. No. 4,970,218. All patents and otherpublications cited herein are hereby incorporated by reference.

It is known that certain compounds within the scope of the presentinvention can induce voltage-dependent blockade of sodium channels invitro and in vivo (Tang et al, 1995; 1998; Tang and Kongsamut, 1996).Voltage-dependent sodium channel blockers act more effectively duringconditions of cellular depolarization. These compounds have little or noeffect on normal neuronal signaling, but allow the blockade of sodiumchannels during seizures, head trauma or ischemia. Many of these agentsare cerebroprotective in animal models of these pathological conditions(Madge et al, 1998).

Without wishing to be bound by theory, potassium channel blockers arealso viable agents for the treatment of neuropathic pain andcytokine-related pain, including arthritic pain. Sweitzer et al (1999)has suggested that microglial activation and cytokine release may play arole in the hyperalgesia following either peripheral inflammation orperipheral nerve injury. Potassium channel blockers, such as 4-AP, havebeen reported to block the activation of rat, mouse and human microglia(Eder, 1998). Pyo et al (1997) have reported that 4-AP can reducenitrite release from activated microglia, indicating that pain behaviorscan be regulated via this mechanism. In addition, 4-AP has been reportedto reduce lipopolysaccahride (LPS)-induced NO production from murinemacrophages (Lowry et al, 1998). The administration of LPS to mice hasalso been used as a model system for the identification ofanti-arthritic efficacy with several different agents with differentmechanisms of action (Mcllay et al, 2001). Several experimental modelswhich involve constriction of the sciatic nerve or the L5 or L6 spinalnerve have been developed to explore neuropathic pain (Bennett and Xie,1988; Seltzer et al, 1990; Kim and Chung, 1992).

SUMMARY OF THE INVENTION

It has now been discovered that compounds of formula I possess potassiumchannel blocking properties. The unique combination of blockingproperties for both the potassium and sodium channels means that thesecompounds are useful as therapeutic agents for the treatment ofdemyelinating diseases or conditions. For example, they are useful intreating MS, SCI, TBI (traumatic brain injury) and stroke. Thesecompounds provide for a safer therapeutic agent than 4-AP because 4-APonly blocks the potassium channel which can lead to the undesirable sideeffects of restlessness, confusion, and seizures. The compounds offormula I are also useful for stroke rehabilitation, the treatment ofbladder irritation and dysfunction, the treatment of visceral,chemokine-induced pain (including arthritic pain) and neuropathic pain.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 illustrates the effect of HP184 on brain damage at 10 and 20mg/kg iv bolus 1 hour after MCA Occlusion—(Example Three).

FIG. 2 shows the behavioral scores of rats after acute treatment withHP184 (ip administration) following spinal cord compression—(ExampleSix).

FIG. 3 shows the behavioral scores of rats after acute treatment withHR184 (po administration) following spinal cord compression—(ExampleSix).

FIG. 4 shows the behavioral scores of rats in the Chronic CrushExperiment 25 days after a mild compression injury—(Example Six).

FIG. 5 shows the normalized difference in scoring for rats with a 25-dayold mild compression injury—(Example Six).

FIG. 6 a shows open field locomotor ability assessment in animals withmoderate compression injury—(Example Six).

FIG. 6 b shows open field locomotor ability assessment data in animalswith moderate compression injury normalized for each rat—(Example Six).

FIG. 7 shows effect of HP184 on demyelinated area at the moderatecompression injury site—(Example Six).

FIG. 8 shows the effects of HP184 on bladder contractionfrequency—(Example 7).

FIG. 9 shows the effect of HP184 on NO production in mice—(Example 8).

FIG. 10 shows the effect of HP184 in a neuropathic pain model—(Example9).

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula I provide a unique combination of blockingproperties for both the potassium and sodium channels. These compoundsare useful for the treatment of Demyelinating Diseases and Conditionssuch as Multiple Sclerosis, Spinal Cord Injury, Traumatic Brain Injuryand Stroke. The compounds are also useful for Stroke Rehabilitation, thetreatment of bladder irritation and dysfunction, the treatment ofvisceral, chemokine-induced pain (including arthritic pain) andneuropathic pain.

wherein

-   -   m is 0, 1 or 2;    -   n is 0, 1 or 2;    -   p is 0 or 1;    -   each R is independently hydrogen, halogen, trifluoromethyl,        C₁–C₆alkyl, C₁–C₆alkoxy, benzyloxy, hydroxy, nitro or amino;    -   each R₁ is independently hydrogen, C₁–C₆alkyl, C₁–C₆alkenyl,        C₁–C₆alkanoyl, halogen, cyano, —C(O)C₁–C₆alkyl,        —C₁–C₆alkyleneCN, —C₁–C₆alkyleneNR′R″ wherein R′ and R″ are each        independently hydrogen or C₁–C₆alkyl,        —C₁–C₆alkyleneOC(O)C₁–C₆alkyl, or —CH(OH)R₄ wherein R₄ is        hydrogen or C₁–C₆alkyl;    -   R₂ is hydrogen, C₁–C₆alkyl optionally substituted with halogen,        hydroxy or benzyloxy, C₁–C₆alkenyl, C₁–C₆alkynyl,        —CO₂C₁–C₆alkyl, or —R₅—NR′R″ wherein R₅ is C₁–C₆alkylene,        C₁–C₆alkenylene or C₁–C₆alkynylene and R′ and R″ are each        independently hydrogen, C₁–C₆alkyl or alternatively the group        —NR′R″ as a whole is 1-pyrrolidinyl; and    -   R₃ is hydrogen, nitro, amino, halogen, C₁–C₆alkoxy, hydroxy or        C₁–C₆alkyl.        Definitions:

-   1) Demyelinating Diseases: As used herein, Demyelinating Diseases    are defined as those diseases in which myelin is the primary target.    They fall into two main groups: acquired diseases and hereditary    metabolic disorders.

Multiple sclerosis (MS) falls under the category of acquired disease. MSusually manifests itself between the 20th and 50th years of life. MSattacks the white matter of the central nervous system. In its classicmanifestation (90% of all cases), it is characterized by alternatingrelapsing/remitting phases—with periods of remission growing shorterover time. Its symptoms include any combination of spastic paraparesis,unsteady gait, diplopia, and incontinence.

The category of Hereditary Metabolic Disorders includes the eightidentified leukodystrophies: metachromatic leukodystrophy, Refsum'sdisease, adrenoleukodystrophy, Krabbe's disease, phenylketonuria,Canavan disease, Pelizaeus-Merzbacher disease and Alexander's disease.The first six are storage disorders. The lack or the malfunctioning ofan enzyme causes a toxic buildup of chemical substances. The etiology ofPelizaeus-Merzbacher and Alexander's diseases, on the other hand,remains unknown.

The clinical course of hereditary demyelinating disorders, which usuallytend to manifest themselves in infancy or early childhood, is tragic.Previously normal children are deprived, in rapid progression, of sight,hearing, speech, and ambulation. The prognosis is death within a fewyears.

-   2) Demyelinating Conditions—As defined herein, a Demyelinating    Condition is a condition that results in deficient myelination. Such    demyelinating conditions include, but are not limited to, spinal    cord injury, traumatic brain injury and stroke.-   3) Spinal Cord Injury (SCI)— As used herein, SCI is defined as an    injury to the spinal cord that results in loss of function such as    mobility or feeling.-   4) Traumatic Brain Injury (TBI)— As used herein, traumatic brain    injury is defined as an injury that results in damage to the brain.    Head injury may occur in one of two ways:    -   A closed head injury occurs when the moving head is rapidly        stopped, as when hitting a windshield, or when it is hit by a        blunt object causing the brain to smash into the hard bony        surface inside the skull. Closed head injury may also occur        without direct external trauma to the head if the brain        undergoes a rapid forward or backward movement, such as when a        person experiences whiplash.    -   A penetrating head injury occurs when a fast moving object such        as a bullet pierces the skull.

Both closed and penetrating head injuries may result in localized andwidespread, or diffuse, damage to the brain. The resulting disabilitiescan include memory loss and emotional disturbance, motor difficulties,including paralysis, and damage to the five senses. In addition, manypatients die from their injuries.

Today, treatment focuses on containing as much damage as possible in the24-hour period following the injury. When someone suffers an injury tothe brain, the resulting devastation extends beyond the initial trauma.A cascade of “secondary damage” ensues. The brain's own immune cellstrigger swelling and fluid buildup, and the injured nerve cells begin tospill out the neurotransmitter called glutamate, which can soonaccumulate to levels that are toxic to the surrounding neurons.

-   5) Stroke rehabilitation—As used herein, stroke rehabilitation is    defined as intervention that results in the recovery functions that    have been lost due to stroke.-   6) Stroke—As defined herein, a stroke occurs when a blood clot    blocks a blood vessel or artery, or when a blood vessel breaks,    interrupting blood flow to an area of the brain. When a stroke    occurs, it kills brain cells in the immediate area. Doctors call    this area of dead cells an infarct. These cells usually die within    minutes to a few hours after the stroke starts. In stroke, measures    of demyelination such as magnetisation transfer ratio (MTR) are    closely related to axonal damage which correlates to motor deficit    (Pendlebury et al, 2000).-   7) Alkyl or alkylene—Unless otherwise stated or indicated, the term    “Alkyl” or “alkylene” means a branched or straight chain alkyl or    alkylene group, as is appropriate to the formula, specified by the    amount of carbons in the alkyl, e.g., C₁–C₆ alkyl means a one, two,    three, four, five or six carbon branched or straight chain alkyl or    alkylene, as the case may be, or any ranges thereof, for example,    but not limited to, C1–2, C1–3, C1–4, C1–5, C2–3, C2–4, C2–5, C2–C6,    C3–C4, C3–5, C3–6, C4–5, C4–6, C5–6, etc.-   8) C₁–C₆alkoxy—Unless otherwise stated or indicated, the term    C₁–C₆alkoxy denotes a straight or branched alkoxy group having from    1 to 6 carbon atoms. Examples of said include methoxy, ethoxy,    n-proxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, t-butoxy and    straight-and branched-chain pentoxy and hexoxy.-   9) Halogen—Unless otherwise stated or indicated, the term halogen    shall mean fluorine, chlorine, bromine or iodine.-   10) C₁–C₆alkanoic acid—Unless otherwise stated or indicated, the    term C₁–C₆alkanoic acid shall mean a carboxylic acid in which the    carboxyl group is attached to hydrogen or an alkyl group of from 1    to 5 carbon atoms.-   11) C₁–C₆alkanoyl—The term C₁–C₆alkanoyl shall mean a group obtained    by removing a hydroxy group from the carboxyl group of a    C₁–C₆alkanoic acid, and thus it includes for instance formyl, acetyl    and the like. The terms alkanoyl, alkenoyl and alkynoyl shall mean    groups obtained by removing a hydroxy group from the carboxyl group    of alkanoic acid, alkenoic acid and alkynoic acid, respectively.    Thus, for instance, linoleyl group derived from linoleic acid is an    example of the term alkenoyl as defined above.-   12) “Pharmaceutically acceptable salts” means either an acid    addition salt or a basic addition salt which is compatible with the    treatment of patients for the intended use.-   13) “Pharmaceutically acceptable acid addition salt” is any    non-toxic organic or inorganic acid addition salt of the base    compounds represented by Formula I or any of its intermediates.    Illustrative inorganic acids which form suitable salts include    hydrochloric, hydrobromic, sulfuric and phosphoric acid and acid    metal salts such as sodium monohydrogen orthophosphate and potassium    hydrogen sulfate. Illustrative organic acids which form suitable    salts include the mono-, di- and tri-carboxylic acids. Illustrative    of such acids are, for example, acetic, glycolic, lactic, pyruvic,    malonic, succinic, glutaric, fumaric, malic, tartaric, citric,    ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic,    phenylacetic, cinnamic, salicyclic, 2-phenoxybenzoic,    p-toluenesulfonic acid and sulfonic acids such as methanesulfonic    acid and 2-hydroxyethanesulfonic acid. Either the mono- or di-acid    salts can be formed, and such salts can exist in either a hydrated,    solvated or substantially anhydrous form. In general, the acid    addition salts of these compounds are more soluble in water and    various hydrophilic organic solvents and which in comparison to    their free base forms, generally demonstrate higher melting points.-   14) “Pharmaceutically acceptable basic addition salts” means    non-toxic organic or inorganic basic addition salts of the compounds    of Formula (I) or any of its intermediates. Examples are alkali    metal or alkaline-earth metal hydroxides such as sodium, potassium,    calcium, magnesium or barium hydroxides; ammonia, and aliphatic,    alicyclic, or aromatic organic amines such as methylamine,    trimethylamine and picoline. The selection criteria for the    appropriate salt will be known to one skilled in the art.-   15) “Stereoisomers” is a general term for all isomers of the    individual molecules that differ only in the orientation of their    atoms in space. It includes mirror image isomers (enantiomers),    geometric (cis/trans) isomers, and isomers of compounds with more    than one chiral center that are not mirror images of one another    (diastereoisomers).-   16) “Patient” means a warm blooded animal, such as for example rat,    mice, dogs, cats, guinea pigs, and primates such as humans.-   17) “Treat” or “treating” means to alleviate symptoms, eliminate the    causation of the symptoms either on a temporary or permanent basis,    or to prevent or slow the appearance of symptoms of the named    disorder or condition.-   18) “Therapeutically effective amount” means a quantity of the    compound which is effective in treating the named disorder, disease    or condition.-   19) “Pharmaceutically acceptable carrier” is a non-toxic solvent,    dispersant, excipient, adjuvant or other material which is mixed    with the active ingredient in order to permit the formation of a    pharmaceutical composition, i.e., a dosage form capable of    administration to the patient. One example of such a carrier is a    pharmaceutically acceptable oil typically used for parenteral    administration.-   20) “Neuropathic Pain” means pain that results from damage to the    nervous system. The nerve damage may be identified or unidentified.    Examples of Neuropathic Pain include post-herpetic neuralgia,    painful diabetic neuropathy, phantom limb pain and central    post-stroke pain.-   21) “Bladder Irritation and Dysfunction” means conditions such as    interstial cystitis and over-active bladder. Overactive bladder is a    distinct medical condition characterized by symptoms including    urinary frequency, urgency, and urge incontinence, the accidental    loss of urine that occurs after the strong sudden urge to urinate.    Diagnosis of overactive bladder is made in the absence of local    pathological or metabolic-related etiologies, with symptoms    attributable to involuntary bladder contractions due to overactivity    of the detrusor muscle. Interstial Cystitus (IC) is a chronic    inflammatory condition of the bladder wall, which frequently goes    undiagnosed.

The compounds of formula I can effectively improve rate and degree ofrecovery in acute spinal cord injury and long-standing spinal cordinjury. They have properties consistent with use-dependent sodiumchannel blockade and voltage-dependent potassium channel blockade invivo. They provide a safer therapeutic than 4-AP. Particularly preferredare compounds wherein R is hydrogen, halogen, trifluoromethyl, orC₁–C₆alkyl; R₁ is hydrogen or C₁–C₆alkyl; R₂ is hydrogen or C₁–C₆alkyl;R₃ is hydrogen, C₁–C₆alkyl or halogen; and p is 0. Further preferredcompounds are those wherein the amino group is attached to the4-position of the pyridine group.

Even more particularly preferred are the compounds of formulas II [alsoknown herein as HP184 orN-(3-fluoro-4-pyridinyl)-N-propyl-3-methyl-1H-indole-1-amine] and III(also known herein as “8183”).

HP184 is very well-tolerated in micromolar brain concentrations one hourafter ip administration of 30 mg/kg HP 184 in rats (Smith et al, 1996).

The unique combination of use-dependent sodium channel blockade andvoltage-dependent potassium channel blockade also differentiates thecompounds of the instant invention from “pure” sodium channel blockerssuch as carbamazepine and phenytoin. These agents have been successfullyused to alleviate “positive” symptoms of MS (painful tonic seizure anddysesthesia). However, they worsen negative symptoms (paralysis andhypesthesia) (Sakurai and Kanazawa, 1999). Compounds of the instantinvention enhance neuronal function due to the fact that they block thepotassium channels. This aids in functional recovery. At present, sodiumchannel blockers are believed useful useful in the treatment of painfulsymptoms and/or as neuroprotective agents. They would not, however, beexpected to enhance rehabilitative efforts.

In treating a patient afflicted with a condition or disorder describedabove, a compound of formula (I) can be administered in any form or modewhich makes the compound bioavailable in therapeutically effectiveamounts, including orally, sublingually, buccally, subcutaneously,intramuscularly, intravenously, transdermally, intranasally, rectally,topically, and the like. One skilled in the art of preparingformulations can determine the proper form and mode of administrationdepending upon the particular characteristics of the compound selectedfor the condition or disease to be treated, the stage of the disease,the condition of the patient and other relevant circumstances. Forexample, see Remington's Pharmaceutical Sciences, 18th Edition, MackPublishing Co. (1990), incorporated herein by reference.

The compounds of Formula I can be administered alone or in the form of apharmaceutical composition in combination with pharmaceuticallyacceptable carriers, the proportion and nature of which are determinedby the solubility and chemical properties of the compound selected, thechosen route of administration, standard pharmaceutical practice andother relevant criteria.

The compounds of the present invention may be administered orally, forexample, in the form of tablets, troches, capsules, elixirs,suspensions, solutions, syrups, wafers, chewing gums and the like andmay contain one or more of the following adjuvants: binders such asmicrocrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch or lactose, disintegrating agents such as alginic acid,Primogel, corn starch and the like; lubricants such as magnesiumstearate or Sterotex; glidants such as colloidal silicon dioxide; andsweetening agents such as sucrose or saccharin may be added or aflavoring agent such as peppermint, methyl salicylate or orangeflavoring. When the dosage unit form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier such aspolyethylene glycol or a fatty oil. Other dosage unit forms may containother various materials which modify the physical form of the dosageunit, for example, as coatings. Thus, tablets or pills may be coatedwith sugar, shellac, or other enteric coating agents. A syrup maycontain, in addition to the present compounds, sucrose as a sweeteningagent and certain preservatives, dyes and colorings and flavors.

The compounds of Formula (I) of this invention may also be administeredtopically, and when done so the carrier may suitably comprise asolution, ointment or gel base. The base, for example, may comprise oneor more of petrolatum, lanolin, polyethylene glycols, bee wax, mineraloil, diluents such as water and alcohol, and emulsifiers andstabilizers.

The solutions or suspensions may also include one or more of thefollowing adjuvants: sterile diluents such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl paraben; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylene diaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials.

The highly lipophilic esters, amides and carbamates of the presentinvention are capable of sustained release in mammals for a period ofseveral days or from about one to four weeks when formulated andadministered as depot preparations, as for example, when injected in aproperly selected pharmaceutically acceptable oil. The preferred oilsare of vegetable origin such as sesame oil, cottonseed oil, corn oil,coconut oil, soybean oil, olive oil and the like, or they are syntheticesters of fatty acids and polyfunctional alcohols such as glycerol orpropyleneglycol.

The depot compositions of the present invention are prepared bydissolving a highly lipophilic ester, amide or carbamate of the instantinvention in a pharmaceutically acceptable oil under sterile conditions.The oil is selected so as to obtain a release of the active ingredientover a desired period of time. The appropriate oil may easily bedetermined by consulting the prior art, or without undue experimentationby one skilled in the art.

The dosage range at which the compounds of Formula I exhibit theirability to act therapeutically can vary depending upon the particulardisease or condition being treated and its severity, the patient, theformulation, other underlying disease states that the patient issuffering from, and other medications that may be concurrentlyadministered to the patient. Generally, the compounds of Formula I willexhibit their therapeutic activities at dosages of between about 0.001mg/kg of patient body weight/day to about 100 mg/kg of patient bodyweight/day.

The following examples are for illustrative purposes only and are notintended to limit the scope of the invention in any way.

EXAMPLE ONE In Vivo Evidence Consistent with Voltage-Dependent SodiumChannel Blockade

Methods: The experimental procedure was based on the method of Bachauβet al (1992). Male CD-1 mice weighing 35–40 g were anaesthetized withchloral hydrate (400 mg/kg). Under an operating microscope, a 3 mmvertical skin incision was made 2 mm behind the right orbit. Thetemporal muscle was deflected and a small craniotomy carried out toexpose the dura. The dura was incised and deflected and the distal partof the right middle carotid artery exposed. The artery was occludedupstream to the main bifurcation by bipolar electroagulation with fineforceps. Infarct volume was measured 24 hours later using 2%triphenyltetrazolium chloride solution. In this experimental paradigm,HP 184 was orally administered to non-fasted mice (10 per group) onehour prior to occlusion. Infarct volume reduction was based oncomparison to 1% acetic acid, vehicle, treated mice. Results are shownin Table 1.

TABLE 1 Neuroprotective activity of HP 184 in the mouse pMCAO strokemodel % infarct volume Dose reduction (mg/kg, po) Time (min) mean sem  1−60 21 ± 3  10 −60 32 ± 11* 10 −60 40 ± 2.5** *= p < 0.05; **= p < 0.01

The neuroprotection observed in a mouse permanent middle corotid arteryocclusion model (PMCAO) is consistent with in vivo voltage-dependentsodium channel blockade at this dose and time.

EXAMPLE TWO Effect of HP184 on EDEMA After Photothrombotic CerebralLesion in the Rat

Rationale/Objective:

Thromboembolic stroke is the third cause of death in the western world.It is caused by a blood clot or disintegrating thrombus either beinggenerated within the cerebral circulation or forming in the heart orlarge vessels and being carried into the cerebral circulation. Bloodflow is then interrupted and an ischaemic lesion develops, with edema,necrosis and apoptosis of tissue. Edema is detrimental because itcompresses the brain, promoting ischaemia, and also cell lysis andmechanical injury. Treatment with HP 184, a joint Na⁺/K⁺ channelblocker, was studied for its effects on this cerebral edema.

Method: Male Sprague Dawley rats (180–200 g bw) were anaesthetized withchloral hydrate (400 mg/kg ip) and placed in a stereotaxic apparatus.The skin was opened to reveal the skull and a cold light (Bioblock 150W) was placed in contact with the right side of the skull forward oflambda. Bengal rose dye (10 mg/kg iv in saline) was administeredintravenously and illumination of the skull started immediately andcontinued for 5 minutes. The skin was then sutured closed over the skulland the animal returned to its cage. Twenty-four hours after thephotothrombotic lesion animals received HP184 made up in 1% tween inwater by intravenous route at 0, 10 or 20 mg/kg body weight in a volumeof 5 ml/kg. One hour later, animals were killed by decapitation andtheir brains removed (see appendix for protocol). Core samples weretaken at the site of the lesion, and contralateral to the lesion, usinga 6 mm diameter cork borer. Water content was determined by wet weightof tissue/dry weight of tissue and edema expressed as % excess water onlesioned sample compared with sample from contralateral hemisphere foreach rat.

Results—Shown in Table 2

TABLE 2 edema (% excess Treatment at 24 h post water) at 25 h lesion npost lesion vehicle 26 4.10 ± 0.12 HP 184 at 10 mg/kg iv 12 3.61 ± 0.23ns HP 184 at 20 mg/kg iv 13 3.20 ± 0.27** stats: ANOVA plus Tukey-Kramer**= p < 0.001

HP 184 demonstrated a significant (22%) reduction of the edema in theright cerebral cortex one hour after iv administration at 20 mg/kg and25 hours after photothrombotic lesion.

EXAMPLE THREE Effect of HP184 on Lesion Size and Neurological FunctionAfter a Transient Focal Cerebral Ischemia in Rats

Rationale/Objective:. In this study HP184 was administered 1 hourpost-ischemia onset in a model of transient focal cerebral ischemia inrats. Parameters measured were lesion size and neurological function.

Methods: Male Sprague-Dawley rats [Iffa Credo, France] weighing about220–240 g were anaesthetized with halothane (1.4%) in a nitrousoxide-oxygen mixture (70:30). Both common carotid arteries (CCAs) wereisolated. The left middle cerebral artery (MCA) exposed via a temporalcraniotomy was occluded with a microclip, and simultaneously the CCAswere occluded for 1 hour. Both body and cerebral temperatures were keptat normothermia. Following surgery animals were returned to their homecages in a room warmed at 24–26° C.

HP184, dissolved in 1% tween (in injectable sterile water), wasadministered at 10 and 20 mg/kg iv 1 hour after ischemia onset, andcontrol rats received the vehicle according to the same protocol. At 24h post-ischemia, a neurological function using a 9 points grading scalewas performed blindly.

GRADING SCALE USED FOR THE NEUROLOGICAL FUNCTION Item Normal scoreDeficit Placing reactions Leg hanging left forepaw 1 0 left 1 0 Visual 10 Grasping reflex left forepaw 1 0 left 1 0 Righting reflex head tiltedleft side 1 0 right 1 0 Abnormal postures Absent Present thorax twisting1 0 left forelimb flexion 1 0 Global neurological score 9

Thereafter rats were killed and brains were removed. Fresh sections werecut with a brain matrix and stained with triphenyl tetrazolium chloride2% at 37° C. for 5 min. The sections were then stored in 10% formalin at4° C. for 24 h. Areas of infarction were measured with an image analyzer(Leica Q500).

Results: Ischemia induced the development of cerebral lesions in boththe cortex and the striatum (See FIG. 1 which illustrates the effect ofHP184 on brain damage at 10 and 20 mg/kg iv bolus 1 hour after MCAOcclusion). HP184 at 10 mg/kg iv significantly reduced the brain lesionsby 41% (p<0.05). This reduction was significant in the cortex (−45%,p<0.05).

EXAMPLE FOUR Measurement of Potassium Channel Blockade

Methods

PC12 cells (ATCC, Rockville, Md.) were grown in Dulbecco's modifiedEagle's media supplemented with 10% fetal bovine serum (GIBCO BRL GrandIsland, N.Y.). Potassium channel currents were measured using standardpatch clamp electrophysiolgy protocols as detailed previously (Rampe etal., 1998).

Results and Discussion

Potassium channel currents were elicited by 200 msec clamp pulses to +40mV from a holding potential of −80 mV. This protocol resulted in asustained outwardly directed current. Application of HP184 (10 μM)reduced the amplitude of this current and enhanced the rate of currentdecay. When current was measured at the end of the pulse, HP184 reducedcurrent amplitude by 75±4% (n=4). The results are consisent with thenotion that HP184 acts as an antagonist of voltage-dependent K⁺ channelsby blocking an activated state.

EXAMPLE FIVE In Vivo Evidence of Enhancement Muscle Function

Objective: The inorganic dye ruthenium red (RuR) has been reported toblock voltage-dependent Ca⁺² current in various cell types, includingmouse sensory neurons (Duchen, 1992), synaptosomes and neuromuscularpreparations (Hamilton and Lundy, 1995; Tapi and Velasco, 1997).Furthermore, RuR blocks release of neurotransmitters in brainsynaptosomes (Meza-Ruiz and Tapia, 1978; Tapia and Meza-Ruiz, 1977) andneuromuscular junction (Alnaes and Rahamimoff, 1975; Person and Kuhn,1979). In vivo, intraperitoneal (ip) administration of RuR causesflaccid paralysis in mice (Tapia et al, 1976) and this effect isantagonized by 4-aminopyridine (4-AP), a voltage-dependent K⁺ channelblocker (Tapia, 1982). Tapia and Velasco (1997) have reviewed theeffects of RuR both in vivo and in vitro, and suggest that RuR interactswith Ca⁺² sites located in the nerve ending membrane. Binding studiesindicate that RuR selectively blocks N-type Ca⁺² channels, and thesechannels regulate the Ca⁺² influx necessary for neurotransmitterrelease. These authors also suggest that ip administration of RuR may bean experimental model of Eaton-Lambert myasthenic gravis syndrome, anautoimmune disease characterized by blockade of Ca⁺² entry and AChrelease due to antibodies that bind to the N-type Ca⁺² channel.Consistent with this possibility, 4-AP has been reported to improvemuscle weakness and restore neuromuscular transmission in patients(Lundh et al, 1977a; 1977b; 1979; McEvoy et al, 1989; Aisen et al,1995).

The ability of both 4-aminopyridine (4-AP) and guanidine to antagonizeRuR-induced flaccid paralysis is possibly due to their ability tofacilitate neurotransmitter release (Lundh, 1978; Lundh and Thesleff,1977; Tapia and Stiges, 1982). In any case, Tapia and coworkers (Tapiaand Stiges, 1982) have reported that RuR blocks the release induced by4-AP in synaptosomes.

In vitro, HP 184 enhances neurotransmitter release by a differentmechanism than does 4-AP. At high concentrations, 4-AP enhances bothelectrically-stimulated and spontaneous release, but these effects arecalcium dependent. In contrast, HP 184 enhances calcium-independentspontaneous neurotransmitter release only (Smith et al, 1993). It hasalso been hypothesized that spontaneous release has a functional role invivo (Smith et al, 1996).

The purpose of the following experiment was to determine if HP 184 and4-AP could antagonize the paralyzing effect of RuR after co-injection.

Method and Results: Groups of 4–5 mice (CD-1; Charles River; 25–35grams) were separately but simultaneously injected ip with ruthenium redand vehicle (1% glacial acetic acid), ruthenium red and 4-AP, orruthenium red and HP 184. The compound known as “8183” was also testedin this paradigm. Starting at 15 minutes after injections, mice wereplaced near a “flagpole” apparatus and their ability to support theirown body weight (ie, to hold on to the flagpole and not fall) wasrecorded. Results were recorded as the number of mice that could supporttheir own body weight versus the total number of mice tested. Theseresults are shown in Table 3. All experiments were performed between 2PMand 4:30PM.

TABLE 3 drug, dose drug, dose 15 min 30 min 45 min RuR, 30 veh 29 out of69 19 out of 69 18 out of 64 mg/kg ip (42%) (27.5%) (30.4%)  0.3 mg/kg4- 22 out of 25 13 out of 25 12 out of 25 AP (88%) (52%) (48%)  0.6mg/kg 4- 12 out of 14  8 out of 14  8 out of 14 AP (85.7%) (57.1)(57.1%)  30 mg/kg HP 15 out of 15 15 out of 15 15 out of 15  184 (100%)(100%) (100%)  10 mg/kg HP 14 out of 15 12 out of 15 11 out of 15  184(93.3%) (80%) (73.3%)  30 mg/kg 13 out of 14 13 out of 14 14 out of 148183 (92.8%) (92.8%) (100%) 100 mg/kg 11 out of 20 10 out of 20 10 outof 20 DPH (55%) (50%) (50%)  30 mg/kg  4 out of 15  3 out of 15  4 outof 15 DPH (26.7%) (20%) (26.7%)  10 mg/kg RIL  9 out of 15  4 out of 15 4 out of 15 (60%) (26.7%) (26.7%)Conclusion:

Both 4-AP (ip) and HP 184 (ip) can antagonize the flaccid paralysisinduced by the ip administration of RuR. This implies that HP 184 isable to enhance neuronal transmission in vivo, possibly via K⁺ channelblockade. It is also possible, as it is for 4-AP, that HP 184 enhancesneuronal transmission, since in vitro brain slice experiments supportincreased brain neurotransmitter release (Smith et al, 1993; 1996).

Doses of the sodium channel blockers diphenylhydaintoin (DPH) andriluzole (RIL) examined in this experimental paradigm were previouslyshown to be neuroprotective in focal ischemia models (Rataud et al,1994; O'Neill et al, 1997). Their lack of effect in this model addssupport to the interpretation that the ability of HP 184 to antagonizeRuR-induced flaccid paralysis is probably not due to in vivo sodiumchannel blockade. This is clinically suggested as well. The negativesymptoms of MS (loss of movement) are often worsened by sodium channelblockers (Sakurai and Kanazawa, 1999).

EXAMPLE SIX Spinal Cord Crush Disease Models

Rationale and Objective: Gruner & Yee (1999) showed that, 25 days afterspinal cord damage, 4-AP enhanced mMEP's following graded spinal cordinjury in rats. Using identical procedures, functional behaviors weremeasured. These behaviors have been shown to correlate with minimalmMEP. The objective of these experiments was twofold:

-   -   1) to determine if HP 184 could attenuate spinal cord        crush-induced motor impairments of moderate intensity if given        acutely and to compare its effectiveness with methylprednisolone        succinate (MPSS), and    -   2) to determine if HP 184 could improve motor function in rats        with long-standing (25 days) spinal cord injury of minor        intensity, and to compare its effect with 4-aminopyridine        (4-AP).        Acute Treatment—i.p. Administration

The spinal cords of female rats were exposed to laminectomy (sham, n=12)or crushed to a diameter of 1.4 mm (5 groups, n=12 each). Normal spinalcord diameter is approximately 2.5 mm. This compression represents amoderate injury characterized by initial open field walking scores of1.5–2.5 in the Open Field Walking Scale. The definitions for the OpenField Walking Scale (OFT) are as follows:

-   0.0 No spontaneous movement-   0.7 Slight movement-   1.0 Movement in hip and/or knee (not ankle)-   1.3 Active movement at hip and knee, not ankle-   1.7 Questionable movement at ankle-   2.0 Movement of the limb in all three major joints-   2.3 Attempts at support-   2.7 Support in stance only-   3.0 Active support, uncoordinated gait-   3.3 Intermittent bouts of coordinated gait-   3.7 Lack of control of ankle or foot, walks on knuckles or on medial    surface of the foot-   4.0 Coordination of forelimbs and hindlimbs in gait-   4.3 Improved hindlimb postural support, abdomen not low to ground-   4.7 One or two toe drags, slight unsteadiness turning at full speed-   5.0 Normal gait and base of support, no loss of balance on fast    turns, no toe drags    Drug Treatment

Within 15 minutes of crush (day 1), rats in HP 184 designated groupsreceived ip injections of 20, 10, 5 or 0 mg/kg in 1% glacial acetic acidvehicle. This administration was repeated on days 2 and 3. MPSS, on theother hand, was administered at 30 mg/kg ip at 15 minutes, 2 hours, 4hours, and 6 hours on day 1 after crush. This MPSS dosing schedule hasbeen described as optimal in the literature, and mirrors the dosingperformed in humans. MPSS is currently the only drug therapy approvedfor human spinal cord injury. FIG. 2 shows the behavioral scores (OFT)of the various treatment groups over time. The normal preoperative scoreis 5. Rate and extent of improvement were significantly different fromvehicle treated rats for both the 20 and 5 mg/kg dose groups. Each pointrepresents the mean plus sem of 8–12 rats.

Acute Treatment—po Administration

Again, the spinal cords of female rats were exposed to laminectomy orcrush to a diameter of 1.4 mm. In HP 184 groups, rats were orallytreated 5–10 minutes prior to crush, and then once a day for days 2 and3. MPSS was dosed as described before. Behavior scores (OFT) are shownin FIG. 3. The normal preoperative score is 5.

Rate and extent of improvement were improved for all doses, includingthe 10 mg/kg group, when compared to the vehicle treated group. Eachpoint represents the mean plus sem of 12 rats.

Chronic Crush Experiment

The spinal cords of female rats were exposed to laminectomy or crush toa diameter of 1.6 mm. This represented a minor injury, and was designedto result in OFT scores of 4.0 after 25 days of no treatment. This waschosen in an attempt to reproduce the same degree of motor impairment asdescribed by Gruner and Yee (1999), who showed 4-AP induced improvementsin hindlimb miniature endplate potential recordings. This procedure andlength of untreated damage has also been shown to result indemyelination. Behavior scores (OFT) are shown in FIG. 4. FIG. 4 showsthe means and standard errors of the groups using the Definitions forthe Open Field Walking Scale described earlier herein.

In this experiment, OFT scores were slightly higher (4.3–4.5), leavingonly a small window for improvement. Using each rat as its own control,consistent improvement was observed after once a day oral dosing of HP184 on Day 26, 27 and 28. Consistent improvement was also observed afteronce a day ip 0.6 mg/kg 4-AP as well. The statistical differences werebased upon the changes for each individual rat (each rat was its owncontrol) using Mann-Whitney U-test. All the behavioral tests on day1,day2 and day3 were performed at 3 hours after gavage. There was no druggiven on day3 (first day started to give drug was day 0). Thestatistical analysis is as follows:

-   20 mg/kg—significant improvement at 3 h to day 3 (p=0.002) compared    to vehicle control-   10 mg/kg—significant improvement at 30 min and 3 h to 12 h (p=0.014)    compared to vehicle control-   3 mg/kg—significant improvement at 30 min to 6 h to day 1 (p=0.0027)    compared to vehicle control-   4-AP—significant improvement at 90 min to 3 h and 12 h to day 2    (p=0.0027) compared to vehicle control

Table 4 illustrates the changes in scoring for each group frompre-dosing to three hours after the third consecutive daily dose.

TABLE 4 Vehicle 4-AP 20 HP 10 HP 3 HP Laminec crush crush crush crushcrush No crush Prior to 4.52 ± 0.04 4.43 ± 0.03 4.54 ± 0.02 4.36 ± 0.014.32 ± 0.05 4.87 ± 0.01 dose 3 hours 4.53 ± 0.03 4.53 ± 0.02 4.60 ± 0.024.44 ± 0.03 4.47 ± 0.04 4.87 ± 0.01 after last dose

FIG. 5 shows the changes in scoring, normalized for each rat. The graphshows the change observed after three consecutive days of dosing (frompre-dosing to three hours after the third consecutive daily dose) witheither 0.6 mg/kg 4-AP (i.p.), 20 or 10 or 3 mg/kg (p.o.). Laminectomyrefers to a sham group. The mean±sem for each group (n=12) is shown inFIG. 5.

Efficacy in Long Standing Spinal Cord Injury

Thirty-five days after a moderate degree of spinal cord injury, oraladministration of 3 mg/kg HP 184 (po) improves motor recovery after asingle dose, and daily dosing for 4 more days resulted in continued andsustained improvement based upon the definitions for the Open FieldWalking Test described earlier herein. 4-AP, at 0.6 mg/kg (ip) wassimilarly effective. A tabular representation of the results from bothchronic spinal cord injury studies (drugs first administered 25 daysafter a mild spinal cord crush and 35 days after a moderate spinal cordcrush) are shown in Table 5.

TABLE 5 Day 25 Day 28 (mild) % possible (mild) 3 hrs after lastimprovement Treatment prior dose Delta (highest score = 5) Control 4.52± .037 4.53 ± .026 .01   2% 4-AP 4.43 ± .030 4.53 ± .023 .10 17.5%* (0.6mpk, ip) HP 184 4.32 ± .016 4.47 ± .035 .15 22.0%* (3 mpk, po) Day 39Day 35 (moderate) (moderate) 3 hrs after last % possible Treatment priordose Delta improvement Control 4.00 ± .074 3.99 ± .057 −.01   −2% 4-AP3.95 ± .084 4.17 ± .047 .22 22.1%* (0.6 mpk, ip) HP 184 3.89 ± .054 4.17± .058  .274 24.8%* (3 mpk, po)

As shown above, HP184 at 3 mg/kg/day by oral gavage from 35 to 41 daysafter moderate crush injury produced significant improvement. It wasnoted in this study that there was more myelin at the site of injury inthe injured spinal cords of rats that received HP184. This data providesevidence consistent with the assertion that HP 184 is either enhancingremyelination or decreasing an ongoing demyelination process.

Further studies were carried out to determine the lowest effective doseof HP184 in the in the moderate chronic (35 days post-injury) crushparadigm in a double blind placebo and positively controlled design. Theeffects of HP184 previously observed at 3 mg/kg, po, were confirmedusing 4AP (0.6 mg/kg, ip) as a positive control. Furthermore, the effectof all treatments on myelin staining was examined histologically.

(1) Behaviourial Assessment

One hundred fifty adult female Wistar rats, 250–300 g weight, obtainedfrom Charles River were housed in the McMaster University HealthSciences Centre (HSC) Central Animal Facilities (CAF) for at least oneweek. During that time they were exposed to the performance testsdescribed below, to ensure they were familiar with them. Rats werehandled daily for 2 weeks prior to surgery.

Rats were anesthetized using isoflurane (3–5%): O2 (1 L/min) in anappropriately equipped surgical suite in the CAF. Temgesic (0.03 mg/kgbody weight, subcutaneously (SQ)) was administered prior to surgery forpain relief. Spinal cords were crushed (compressed) with a 3.5 mm widemodified coverslip forceps (Blight 1991, procedure revised by Rathbonelaboratory). The forceps were closed to 1.4 mm for 15 sec, whichproduced injury level equivalent to the mid-level (moderate) outcome onthe Gruner scale (1996). The compression injury was otherwise performedaccording to the procedure described by Blight (1991).

The animals were observed to determine pain behaviours, for presence ofurinary tract infections or urinary retention. Pain was treated withTynenol (0.8 mg/10 gm body weight orally).

To prevent the urinary infection, Septra (Trimethoprin-Sulfamethoxazole)was given orally (4.5 ml in 300 ml water) 1 day pre- and 5 dayspost-operation, and were treated with manual bladder expression. In thecase of infections, i.e. any urinary tract infection, indicated bycloudy or bloody urine, Baytril (enrofloxacin, 7 mg/kg b.w.) wasinjected subcutaneously (SQ) twice a day.

Changes in locomotor behaviour and segmental reflexes were assessed upto 5 weeks post injury. Animals were tested in an open field walkingtask, hind limb placement and foot orientation. The animals wereevaluated on days 2, 7, 14, 21, 28 and 35 after surgery. By 35 daysafter surgery, almost no further spontaneous recovery occurs. Thereforetreatment began on day 35.

HP184 was dissolved in sterilized (autoclaved) deionized reverse-osmosiswater acidified with glacial acetic acid (0.1 ml acid per 10 ml ofwater). 4-AP (Sigma, molecular weight 94.12; Jankowska E. et al., 1982;Gruner et al., 1999) was dissolved in physiological saline (0.6 mg/kgb.w.) and was administered by i.p. injection. One group of rats (vehiclecontrol-1) received by oral gavage vehicle. Behaviourial testing wasdone immediately prior to receiving the gavage and at 3 hoursthereafter. Then, the rats were scarified on day 35. All the other ratsreceived either HP184 by oral gavage (0.3, 1, or 3 mg/kg bw depending onthe group) or 4-AP (0.6 mg/kg, i.p.) or vehicle (vehicle control-2) oncea day on the 35 to 42 days after surgery. On these days behaviourialtests were done immediately prior to receiving the gavage at 3 and 24hours thereafter. Then, the rats were perfused on day 43 after the lastbehaviourial testing.

Video recording of the behaviourial testing using Hi-8, was done on days35 to 43 after surgery.

Statistical analyses were performed on a Macintosh computer usingGB-Stat ppc 6.5.2. The behavioral scores were analyzed by theKruskal-Wallis nonparametric analysis of variance (ANOVA). Post hoccomparisons were made using Mann-Whitney U tests.

The overview recovery of open field locomotor ability was assessed bythe mean OFT scores for each groups, which are shown in FIGS. 6 a and 6b. These results show that the performance of animals treated with HP184or 4-AP was significantly different from that of control animalsreceiving vehicle. ANOVA for repeated measures shows treatment effect(p<0.01) on days 35–42.

The results show that both 4-AP and HP184 have beneficial effects,improving behavioral testing after moderate chronic spinal compression.Although all three concentrations of HP184 had beneficial effects, the 3mg/kg of HP184 produced the best recovery of locomotor function therebyconfirming the effects of HP184 observed previously at this dose. Theseresults also indicate that the lowest (0.3 mg/kg) concentration of HP184 may not be the lowest effective dose of HP184 in this paradigm.

Histological Study of Spinal Cords

A study to test whether the treatment with HP184 affected the amount ofmyelin in rats with moderate long term spinal cord crush injury whenadministered long after spinal cord injury.

The spinal cords from rats described above in the assessment were usedfor this study.

On postoperative day 21, the experimental subjects were deeplyanesthetized with sodium pentobarbital (50–60 mg/kg body weight, i.p.)and perfused transcardically—first with 100 mL 0.05M phosphate buffedsaline (PBS) containing 0.1% heparin, followed by 300–500 mL of 4%paraformaldehyde (PFA). Segments T9 to L1 of the spinal cords were takenout, then cryo-protected in 30% sucrose solution and frozen at −70° C.in 10.24% polyvinyl alcohol and 4.26% polyethylene glycol.

A segment of each cord including the lesion site plus 10 mm rostral andcaudal to the lesion site was embedded in Tissue Tek medium. Serialsections were cut longitudinally at 20 μm intervals on a cryostat. Everythird section was stained with luxol fast blue for myelin. Theevaluation was performed by observers blinded as to treatment, on codedslides. Sections were examined under a light microscope for the extentof demyelination (the area without luxol fast blue staining).

For determinations of the maximal demyelinated area of the cord, thewhole section was digitized on photographs using a Zeiss microscope. Theextent of demyelination was measured at the lesion center using acomputerized Bioquant BQ-TCW98 image analysis progrem by an investigatorwho was blind to treatment group.

Statistical analysis was performed on a Macintosh computer using GB-Statppc 6.5.2. The histological results were analyzed by the Kruskal-Wallisnonparametric analysis of variance (ANOVA). Post hoc comparisons weremade using Mann-Whitney U tests.

The extent of demyelination for the six experimental groups (0.3, 1, or3 mg/kg bw depending on the group or 4-AP 0.6 mg/kg or vehicle control 1and 2) is shown in FIG. 7. The bars represent the number of pixels ofdemyelinated area at the crush center. (**P<0.001, *P<0.05,Kruskal-Wallis nonparametric analysis of variance (ANOVA)) Thequantitative results show that the cords from HP184 or 4-AP treatedanimals had significantly greater myelinated area than that of salinecontrols. That is, the cords from animals which received vehicleinjections had a significantly greater demyelinated area than that ofeither HP184 or 4-AP treated animals.

The histological analysis showed that both HP184 (at all threeconcentrations) and 4-AP have beneficial effects on myelination, whichwas consistent with the behavioral testing results. Of those groups,animals treated with 3 mg/kg of HP 184 showed the least demyelination.Therefore, 4-AP or HP184 appears capable of enhancing re-myelination ata stage long after spinal cord injury. It is improbable that the datasimply represent a reduction in the rate of loss of myelin, since therewas no difference in the extent of demyeliation in the two controlgroups, control 1 and control-2, evaluated at the beginning and end ofthe experiment.

EXAMPLE 7 The Effect of Intravenous HP-184 on Bladder Irritation in theRat

This experiment shows the effect of intravenous HP184 in the KCl modeloutlined by Fraser et al (2001). Fraser et al combined protamine sulfatetreatment, thought to breakdown urothelial umbrella cell barrierfunction, and physiologic urine concentrations of KCl (500 mM). Theeffects of intravenous HP-184 were compared to vehicle alone (n=4/group)in a cumulative dose-response study in urethane anesthetized rats withacute bladder irritation. Continuous open cystometry, which measures thefilling and emptying of the bladder during continuous infusion, wasutilized to determine the effect of the drug on bladder irritation. Whenthe bladder is irritated, it contracts more frequently during the samefilling rate due to sensitization of C-fiber afferent nerves. FIG. 8illustrates the dose-dependent decrease in bladder contraction frequencyfrom pre-administration irritation values compared to the effects ofvehicle alone. Analysis of Variance for Repeated Measures indicates thatwhile vehicle alone had no effect, HP-184 significantly decreasedbladder contraction frequency in irritated bladders in a dose-dependentfashion (P=0.0019).

EXAMPLE 8 The Effect of HP184 on No Production in Mice

Mice were injected with 30 mg/kg HP 184 (ip) 30 minutes prior to LPS (3mg/kg, ip). Mice were sacrificed 5 hours after LPS injection, and plasmacollected. Nitrate levels were determined by the Griess assay. Groupswere composed of 9–10 mice each. As shown graphically in FIG. 9, HP184inhibits NO production. After one-way ANOVA, only LPS treatment wasfound to be significantly different (p<0.01) from vehicle treatment.

EXAMPLE 9 HP184 in a Neuropathic Pain Model

Adult male Sprague-Dawley rats received unilateral constriction of theL6 nerve to produce chronic nerve injury. Following recovery fromsurgery (3–7 days post operative) animals were tested for paw withdrawalthreshold to mechanical stimuli applied to the affected paw. This wasdetermined by the application of calibrated von Frey monofilaments tothe plantar surface of each hindpaw. Only animals with a 50% decrease inwithdrawal threshold in the ligated paw were employed in the study, andwere randomly assigned to one of 6 groups: three groups receiving one ofthree doses of HP 184 (0.3, 3 and 20 mg/kg, po), a fourth groupreceiving a single dose of another compound referred to MDL (10 mg/kg,ip), a fifth group receiving gabapentin (90 mg/kg, sc), and a sixthgroup receiving vehicle only. Behavioral testing occurred 45 minutesfollowing the gabapentin (90 mg/kg, sc), and 3 hours following the HP184, MDL, and vehicle. A difference score between the ligated andnon-ligated paw withdrawal thresholds is calculated for each animal, andthese differences were subjected to ANOVA with group as the main factor.The results are shown in FIG. 10. The graph shows the Mean (+/−SEM)difference of left (ligated) minus right (normal) paw withdrawalthreshold before and after the first drug administration (acute phase ofstudy). Statistical analysis reveals a dose-response attentuation of L5ligation mechanical hyperlagesia by HP184 20 mg/kg and a clear reversalof hyperalgesia by gabapentin 90 mg/kg. Analysis was between/withinrepeated measures ANOVA. This was followed by post-hoc comparison (LSD)on the group X time interaction term to exam pre versus post drugwithdrawal threshold values.

-   Group: F(5,43)=8.18, p<0.001-   Time: F(1,43)=47.34, p<0.001-   Group X time: F(5,43)=9.25, p<0.001

In vehicle treated animals, there is a large difference in mechanicalwithdrawal thresholds between the two paws.

REFERENCES

-   Agoston S, Bowman W C, Houwertjes M C, Rodger I W, Savage A O.    Direct action of 4-aminopyridine on the contractility of a    fast-contracting muscle in the cat. Clin Exp Pharm Physiol 1982; 9:    21–34.-   Aisen M L, Sevilla D, Gibson G, Kutt H, Blau A, Edelstein L, Hatch J    and Blass J (1995) 3,4-Diaminopyridine as a treatment for amyotropic    lateral sclerosis. J Neurol Sci. 129:21–24.-   Alnaes E and Rahaminoff R (1975) On the role of mitochondria in    transmitter release from motor nerve terminals. J. Physiol (Lond)    248:285–306.-   Backhauβ C, Karkoutly C, Welsch M, and Krieglstein J (1992): A mouse    model of focal cerebral ischemia for screening neuroprotective drug    effects. J Pharmacological Meth. 27:27–32.-   Ball A P, Hpokinson R B, Farrell I D (1979): Human botulism caused    by Clostridium Botulinum type E: the Birmingham outbreak Q. J. Med.    48–473–491.-   Behrmann D L, Bresnahan J C, Beattie M S, Shah B R. Spinal cord    injury produced by consistent mechanical displacement of the cord in    rats: behavioral and histologic analysis. J. Neurotrama, 9:197–217,    1992.-   Bennett G J and Xie Y K (1998) A peripheral mononeuropathy in rat    produces disorders of pain sensation like those seen in man. Pain.    33:87–107.-   Bever C T (1996) Aminopyridines in Handbook of Multiple Sclerosis,    ed S D Lick, Marcel Dekker, pp 429–42.-   Bever C T, Jr., Young D, Anderson P A, Krumholz A, Conway K, Leslie    J, Eddington N, Plaisance K I, Panitch H S, Dhib-Jalbut S. The    effects of 4-aminopyridine in multiple sclerosis patients: results    of a randomized, placebo-controlled, double-blind,    concentration-controlled, crossover trial. Neurol 1994; 44:    1054–1059.-   Blight A R and DeCrescito V. Morphometric analysis of experimental    spinal cord injury in the cat: the relation of injury intensity to    survival of myelinated axons. Neuroscience 1986: 19:321–41.-   Blight A R. Morphology of chronic spinal cord injury in the cat:    Analysis of myelinated axons by line-samping. Neuroscience,    10:521–543, 1983.-   Blight A R. Morphometric analysis of a model of spinal cord injury    in guinea pigs, with behavioral evidence of delayed secondary    pathology. J Neurol Sci, 103: 156–171, 1991.-   Bostock H, Sherratt R M, Sears T A. Overcoming conduction failure in    demyelinated nerve fibres by prolonging action potentials. Nature    1978; 274: 385–387.-   Bostock H, Sears T A, Sherratt R M. The effects of 4-aminopyridine    and tetraethylammonium ions on normal and demyelinated mammalian    nerve fibres. J Physiol(Lond) 1981; 313: 301–315.-   Bunge R P, Puckett W R, Bercerra J L, Marcillo A, Quencer R M.    Observations on the pathology of human spinal cord injury. A review    and classification of 22 new cases with details from a case of    chronic cord compression with extensive focal demyelination. In:    Seil F J, ed. Advances in neurology, vol 59, New York: Raven Press,    1993:75–89.-   Davis F A, Stefoski D, Rush J. Orally administered 4-aminopyridine    improves clinical signs in multiple sclerosis. Ann Neurol 1990; 27:    186–192.-   Duchen, M R (1992) Ca+2-dependent changes in the mitochondrial    energetics in single dissociated mouse sensory neurons. Biochem J.    283:41–50.-   Eder C (1998) Ion channels in microglia (brain macrophages) Am. J.    Physiol. 275 (Cell Physiol. 44):C327–C342.-   Fraser M O, Chuang Y, Lavelle J P, Yoshimura N, de Groat W C,    Chancellor M B (2001) a reliable, nondestructive animal model for    interstitial cystitus: intravesical low-dose protamine sulfate    combine with physiological concentrations of potassium chloride.    Urology 57(Suppl 1): 112-   Gruner J A and Yee A K (1999) 4-Aminopyridine enhances motor evoked    potentials following graded spinal cord compression injury in rats.    Brain Res. 816:446–56.-   Gruner J A, Wade C K, Menna G and Stokes B T. Myoelectric evoked    potentials Versus locomotor recovery in chronic spinal cord injured    rats. J. neurotrauma, 10:327–347, 1993.-   Gruner J A, Yee A K, Blight A R. Histological and functional    evaluation of experimental spinal cord injury: evidence of a    stepwise response to graded compression. Brain Res., 729:90–101,    1996.-   Gruner J A, Yee A K. 4-Aminopyridine enhances motor evoked    potentials following graded spinal cord compression injury in rats.    Brain Res. Jan 23; 816(2):446–56, 1999.-   Hamilton, M G and Lundy P M (1995) Effect of ruthenium red on    voltage-sensitive Ca+2 channels. J PET 273:940–947.-   Hayes K C, Blight A R, Potter P J, Allatt R D, Hsieh J T, Wolfe D L,    Lam S, Hamilton J T. Preclinical trial of 4-aminopyridine in    patients with chronic spinal cord injury. Paraplegia 1993; 31:    216–224.-   Hayes K C, Potter P J, Wolfe D L, Hsieh J T, Delaney G A, Blight    A R. 4-Aminopyridine-sensitive neurologic deficits in patients with    spinal cord injury. J Neurotrauma 1994; 11: 433–446.-   Hirsh J K, Quandt F N. Aminopyridine block of potassium channels in    mouse neuroblastoma cells. J Pharmacol Exp Ther 1993; 267: 604–611.-   Hockfield S. Carolson S, Evans C, et al. Selected methods for    antibody and nuceic acid probes. USA: Cold Sprint Harbour Laboratory    Press, p. 125–130, 1993.-   Jankowska. E, Lundberg A., Rudomin P. and Sykova E. Effects of    4-Aminopyridine on synaptic transmission in the cat spinal cord.    Brain Research, 240:117–129, 1982.-   Jones R E, Heron J R, Foster D H, Snelgar R S, Mason R J. Effects of    4-aminopyridine in patients with multiple sclerosis. J Neurol Sci    1983; 60: 353–362.-   Kerasidis H, Wrathall J R and Gale K. Behavioral assessment of    functional deficit in rats with contusive spinal cord injury. J.    Neurosci. Methods, 20:167–179, 1987.-   Lowry M A R, Goldbert J I and Belosevic M (1998) Induction of nitric    oxide (NO) synthesis in murine macrophages requires potassium    channel activity. Clin Exp Immunol 111:597–603.-   Lundh H (1978) Effects of 4-aminopyridine on neuromuscular    transmission. Brain Res. 153:307–318.-   Lundh H and Thesleff S (1977) The mode of action of 4-aminopyridine    and guanidine on transmitter from motor nerve terminals. Eur. J.    Pharmacol. 42:411–12.-   Lundh H, Nilsson O, Rosen I. 4-aminopyridine—a new drug tested in    the treatment of Eaton-Lambert syndrome. J Neurol Neurosurg Psychiat    1977; 40: 1109–1112.-   Lundh H, Leander S, Thesleff S (1977): Antagonism of the paralysis    produced by botulinum toxin in the rat. J. Neurol. Sci. 32:29–43.-   Kim S H ans Chung J M (1992) An experimental model for peripheral    neuropathy produced by segmental spinal nerve ligation in the rat.    Pain 50:355–363-   Madge D J (1998): Sodium channels: recent developments and    therapeutic potential, In Annual Reports in Medicinal Chemistry,    Volume 33 (Bristol J A Editor in chief, Academic Press, San Diego),    pp 51–60.-   McEvoy K M, Windebank A J, Daube J R and Low P (1989):    3,4-Diaminopyridine in the treatment of Lambert-Eaton myasthenic    syndrome (N. Engl. J. Med. 321:1567–71.-   Mcllay L M, Halley F, Souness J E McKenna J, Benning V, Birrell M,    Burton B, Belvisi M, Collis A, Constan A, Foster M, Hele D, Jayyosi    Z, Kelley M, Maslen C, Miller G, Ouldelhkim M C, Page K, Phipps S,    Pollock K, Porter B, Ratcliffe A J, Redford E J, Webber S, Slater B,    Thybaud V, Wilsher N (2001) The discovery of RPR 200765A, a p38 MAP    kinase inhibitor displaying a good oral anti-arthritic efficacy.    Bioorg Med Chem 9:537–54.-   Meza-Ruiz G and Tapia R (1978) [3H]GABA release in synaptosomal    fractions after intracranial administration of ruthenium red. Brain    Res. 154:163–166.-   O'Neill M J, Bath C P, Dell C P, Hicks C A, Gilmore J, Ambler S J,    Ward M A, Bleakman D (1997): Effects of Ca2+ and Na+ channel    inhibitors in vitro and in global cerebral ischaemia in vivo. Eur J    Pharmacol 332(2):121–31 RIL 10 mg/kg reference-   Pendlebury S T, Lee M A, Blamire A M, Styles P, and Matthews P    M (2000) Correlating magnetic resonance imaging markers of axonal    injury and demyelination in motor impairment secondary to stroke and    multiple sclerosis. Magn. Reson. Imaging 18:369–78.-   Person R J and Kuhn J A (1979) Depression of spontaneous and    ionophore-induced transmitter release by ruthenium red at the    neuromuscular junction. Brain Res. Bull 4:669–674.-   Potter P J, Hayes K C, Hsieh J T, Delaney G A, Segal J L. Sustained    improvements in neurological function in spinal cord injured    patients treated with oral 4-aminopyridine: three cases. Spinal Cord    1998a; 36:147–155.-   Potter P J, Hayes K C, Segal J L Hsieh J T, Brunnemann S R, Delaney    G A, Tierney D S and Mason D (1998b): Randomized double-blind    crossover trial of fampridine-SR (sustained release 4-aminopyridine)    in patients with incomplete spinal cord injury. J. Neurotrauma    15:837–49.-   Pyo H, Chung S, Jou I, Gwag B and Joe E H (1997) Expression and    function of outward K⁺ channels induces by lipopolysaccharide in    microglia. Mol Cells 7:610–614.-   Qiao J, Hayes K C, Hsieh J T, Potter P J, and Delaney G A (1997):    Effects of 4-aminopyridine on motor evoked potentials with spinal    cord injury. J Neurotrauma 14:135–49.-   Rampe, D., Murawsky, M. K., Grau, J. and Lewis, E. W. The    antipsychotic agent sertindole is a high affinity antagonist of the    human cardiac potassium channel HERG. J. Pharmacol. Exp. Ther. 286:    788–793, 1998.-   Rataud J, Bebarnot F, Mary V, Pratt J and Stutzmann J M (1994):    Comparative study of voltage-sensitive sodium channel blockers in    focal ischaemia and electric convulsions in rodents. Neuro Sci Lett.    172:19–23.-   Sakurai M and Kanazawa I (1999) Positive symptoms in multiple    sclerosis: their treatment with sodium channel blockers, lidocain    and mexiletine. J Neurol. Sci. 162:162–168.-   Saruhashi Y and Young W. Effect of mianserin on locomotory function    after thoracic spinal cord hemisection in rats. Expl Neurol.,    129:207–216, 1994-   Savage A O. A comparison of the effects of 4-dimethylaminopyridine    and 4-aminopyridine on isolated cardiac and skeletal muscle    preparations. Arch Internat Pharmacodynam Therapie 1985; 273:    262–276.-   Schwid S R, Petrie M D, McDermott M P, Tierney D S Mason D H and    Goodman A D (1997): Quantitative assessment of sustained release    4-aminopyridine for symptomatic relief of multipule sclerosis.    Neurology 48:817–21.-   Segal J L, Pathak M S, Hernandez J P, Himber P L, Brunnemann S R and    Charter R S (1999): Safety and efficacy of 4-aminopyridine in humans    with spinal cord injury: A long-term, Controlled Trial.    Pharmacotherapy 19:713–723.-   Seltzer Z, Dubner R and Shir Y (1990) A novel behavioral model of    neuropathic pain disorders produced in rats by partial sciatic    nerveinjury. Pain 43:205–218.-   Sherratt R M, Bostock H, Sears T A. Effects of 4-aminopyridine on    normal and demyelinated mammalian nerve fibres. Nature 1980; 283:    570–572.-   Shi R, Blight A R. Differential effects of low and high    concentrations of 4-aminopyridine on axonal conduction in normal and    injured spinal cord. Neurosci 1997; 77: 553–562.-   Smith, C. P., A. T. Woods, Corbett, R., S. M. Chesson, G. M.    Bores, W. W. Petko, J. E. Roehr and S. Kongsamut. Serotonergic    activity of HP 184: Does spontaneous release have a role?    Neurochemical Research 21:573–583, 1996.-   Smith, C. P., L. R. Brougham, F. P. Huger, L. Davis, J. T. Klein    and R. C. Effland. H P 184    [N-(n-propyl)-N-(3-fluroro-4-pyridinyl)-1H-3-methylindol-1-amine    hydrochloride]: In vitro spontaneous release of acetylcholine (ACh)    and norepinephrine (NE). Drug Dev. Res. 30:203–212, 1993.-   Stefoski D, Davis F A, Faut M, Schauf C L. 4-Aminopyridine improves    clinical signs in multiple sclerosis. Ann Neurol 1987; 21: 71–77.-   Sweitzer S M, Colburn R W, Rutkowski M and DeLeo J A (1999) Acute    peripheral inflammation induces moderate glial activation and spinal    IL-1    expression that correlates with pain behavior in the rat. Brain Res.    829:209–221.-   Tang L and Kongsamut S (1996) Frequency-dependent inhibition of    neurotransmitter release by besipirdine and H P 184. Eur J Pharmacol    300:71–74.-   Tang, L., Huger, F. P., Klein, J. T., Davis, L., Martin, L.,    Shimshock, S., Effland, R. C., Smith, C. P. and Kongsamut, S. (1998)    4-Aminopyridine derivatives: A family of novel modulators of    voltage-dependent sodium-channels. Drug Dev. Res., 44:8–13.-   Tang L., C. P. Smith and S. Kongsamut. Besipirdine inhibits effects    of veratridine at the voltage dependent sodium channel. Br J.    Pharmacol 116:2468–2472, 1995.-   Tapia R and Velasco I (1997) Ruthenium red as a tool to study    calcium channels, neuronal death and the function of neural    pathways. Neurochem Int 30:137–147.-   Tapia R and Meza-Ruiz G (1977) Inhibition by ruthenium red of the    calcium-dependent release of [3H]GABA in synaptosomal fractions.    Brain Res. 126:160–166.-   Tapia R, Meza-Ruiz G, Duran L and Drucker-Colin R D (1976)    Convulsions or flaccid paralysis indued by ruthenium red depending    on route of administration. Brain Res. 116:101–109.-   Tapia R and Stiges M (1982) Effect of 4-aminopyridine on transmitter    release in synaptosomes. Brain Res. 250:291–9.-   Tapia R (1982) Antagonism of the ruthenium red-induced paralysis in    mice by 4-aminopyridine, guanidine and lanthanum. Neurosci Lett    35:615–623.-   Targ E F, Kocsis J D. 4-Aminopyridine leads to restoration of    conduction in demyelinated rat sciatic nerve. Brain Res 1985; 328:    358–361.-   Targ E F, Kocsis J D. Action potential characteristics of    demyelinated rat sciatic nerve following application of    4-aminopyridine. Brain Res 1986; 363: 1–9.-   van Diemen H A, Polman C H, van Dongen T M, van Loenen A C, Nauta J    J, van Walbeek H K, Koetsier J C. The effect of 4-aminopyridine on    clinical signs in multiple sclerosis: a randomized,    placebo-controlled, double-blind, cross-over study. Ann Neurol 1992;    32: 123–130.-   van Diemen H A, Polman C H, van Dongen M M, Nauta J J, Strijers R L,    van Loenen A C, Bertelsmann F W, Koetsier J C. 4-Aminopyridine    induces functional improvement in multiple sclerosis patients: a    neurophysiological study. J Neurol Sci 1993; 116: 220–226.-   Yamaguchi S and Rogawski M A (1992): Effects of anticonvulsant drugs    on 4-aminopyridine-induced seizures in mice. Epilepsy Res. 11:9–16.    Patents:-   Effland R C, Klein J T, Davis K L Olsen G E; U.S. Pat. No. 4,970,218    entitled “N-(Pyridinyl)-1H-indol-1-amines”.-   Hansebout R R and Blight A R; U.S. Pat. No. 5,545,648 entitled “Use    of 4-aminopyridine in the reduction of chronic pain and spasticity    in a spinal cord injured patient”.-   Hansebout R R and Blight A R; WO 94/14439 entitled “The use of    4-aminopyridine in the treatment of a neurological condition”.-   Huger, F. P., Kongsamut, S., C. P. Smith & L. Tang. U.S. Pat. No.    5,776,955 entitled “Use of unsubstituted and substituted    N-(pyrrol-1-yl) pyridinamines as anticonvulsant agents”.-   Kongsamut, S., C. P. Smith & A. T. Woods; U.S. Pat. No. 5,356,910    entitled “Use of N-(Pyridinyl)-1H-indol-1-amines for the Treatment    of Obsessive Compulsive Disorder”.-   Kongsamut, S., C. P. Smith & A. T. Woods; U.S. Pat. No. 5,356,910    entitled “Use of N-(Pyridinyl)-1H-indol-1-amines for the preparation    of a medicament for the treatment of obsessive-compulsive    disorders”.-   Masterson J G and Myers M; U.S. Pat. No. 5,370,879 entitled    “Formulations and their use in the treatment of neurological    diseases”.-   Masterson J G and Myers M; U.S. Pat. No. 5,580,580 entitled    “Formulations and their use in the treatment of neurological    diseases”.-   Masterson J G and Myers M; U.S. Pat. No. 5,540,938 entitled    “Formulations and their use in the treatment of neurological    diseases”.-   Wurtman R J and Buyukysal R; WO 89/09600 entitled “Method and    composition for treating neurological disorders”.

1. A method of treating Bladder Irritation said method comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of formula I

wherein m is 0, 1 or 2; n is 0, 1 or 2; p is 0 or 1; each R isindependently hydrogen, halogen, trifluoromethyl, C₁–C₆alkyl,C₁–C₆alkoxy, benzyloxy, hydroxy, nitro or amino; each R₁ isindependently hydrogen, C₁–C₆alkyl, C₁–C₆alkenyl, C₁–C₆alkanoyl,halogen, cyano, —C(O)C₁–C₆alkyl, —C₁–C₆alkyleneCN, —C₁–C₆alkyleneNR′R″wherein R′ and R″ are each independently hydrogen or C₁–C₆alkyl,—C₁–C₆alkyleneOC(O)C₁–C₆alkyl, or —CH(OH)R₄ wherein R₄ is hydrogen orC₁–C₆alkyl; R₂ is hydrogen, C₁–C₆alkyl optionally substituted withhalogen, hydroxy or benzyloxy, C₁–C₆alkenyl, C₁–C₆alkynyl,—CO₂C₁–C₆alkyl, or —R₅—NR′R″ wherein R₅ is C₁–C₆alkylene,C₁–C₆alkenylene or C₁–C₆alkynylene and R′ and R″ are each independentlyhydrogen, C₁–C₆alkyl or alternatively the group —NR′R″ as a whole is1-pyrrolidinyl; and R₃ is hydrogen, nitro, amino, halogen, C₁–C₆alkoxy,hydroxy or C₁–C₆alkyl or a pharmaceutically acceptable salt thereof. 2.The method of claim 1 wherein the compound has the following formula: